The gypsy moth (Lepidoptera: Lymantriidae, Lymantria dispar L.) is one of the most feared defoliators of more than three hundred of man's most valued plants, shrubs and trees throughout much of the world. In particular, the gypsy moth attacks oak, aspen, other deciduous trees, and some conifers, causing a significant economic problem throughout the world. Hundreds of millions of dollars have been spent to suppress the damage from this major pest. In addition, the nuisance from large numbers of gypsy moth caterpillars and dropping frass (i.e., feces) severely reduces the aesthetic and recreational values of individual shade trees as well as forests.
Two possible methods to control gypsy moths include injection of systemic insecticides into susceptible plants, and/or application of a bacterial pathogen (Bacillus thuringiensis var. Kurstaki) of gypsy moths. Both methods are costly. Systemic insecticide injection into valued trees costs approximately $6/5 m.sup.3 of trunk. Aerial application of the commercially available bacterial pathogen of the insect costs approximately $5.25-7.00/acre. However, both the systemic insecticide and the bacterium are toxic to other life in the environment, and provide inconsistent suppression of gypsy moth.
There has consequently been a significant effort to develop cost-effective antixenosic and/or antibiotic substances specifically targeted to the gypsy moth, which substances are nontoxic to other life. Antixenosis is a condition which causes an insect to not prefer, or to prefer less, a plant as host, and/or to alter its behavior in ways which decrease its feeding, oviposition and/or sheltering. Antibiosis is a plant condition in which metabolic inhibition and/or toxicity occurs.
In most bioassays testing potential insect repellents and insecticides, duration of the experiment and chemical concentration seem to be major factors in determining whether antixenosis and/or antibiosis will occur. In shorter experiments, antixenosis is more likely; in longer ones the effect is often a combination of antibiosis and antixenosis. Lower concentrations frequently yield antixenosic, but not antibiotic effects.
Researchers have long observed that certain plants repel pestilential insects, including L. dispar. For example, it has been observed that leaf extractables of Pinus silvestris L. and Ilex aquifolium L., and the specific monoterpenes: .alpha.-pinene, .beta.-pinene, 3-carene and camphene have phagodeterrent effects on L. dispar. When applied on standardized styrofoam disks, all chemical treatments showed a significant reduction in larval feeding. The researchers hypothesized that leaves of P. silvestris, being rich in etheric oils, also may produce volatile deterrents. Meisner, J. and Skatulla, U., Phytoparasitica 3: 19-26. (1975).
The antifeedant effects of Kalmia latifolia L. leaf extractables have also been tested on L. dispar larvae. Ten antifeedant diterpenes were isolated and characterized from the ethyl acetate-extractable fraction of the ethanolic extractables. Kalmitoxin-I, kalmitoxin-IV and grayanotoxin-III were shown to be the more active. The level of activity was determined as dryfrass weight per treatment, and was reported as a percentage of feeding reduction by a treatment as compared to the control (red oak). El-Naggar, S. F., Doskotch, R. W., Odell, T. M., and Girard, L., J. Nat. Prod. 43:617-631 (1980).
A sesquiterpene alcohol, nerolidol, isolated from the hexane extractables of the Melaleuca leucadendron L. leaf also has been shown to act as a feeding deterrent. Doskotch, R. W., Cheng, H.-Y., ODell, T. M., and Girard, L., J. Chem. Ecol. 6:845-851 (1908). Although nerolidol is not considered to be a highly potent compound, its high concentration in the plant may explain its observed activity. Related alcohols such as geraniol (a monoterpenoid alcohol present in the genus Jasminum of the family Oleaceae) and farnesol, showed more activity than nerolidol. In contrast, the simpler isoprene-structured compounds, 2-methyl-3-buten-2-ol and t-amyl alcohol, were inactive. Gibbs, R. D., McGill-Queen's University Press, Montreal (1974).
It has also been found that the feeding deterrency of Catalpa speciosa Warder leaf extractables is due to the synergistic effects of several compounds. Fractionation of the active ethyl acetate extractables yielded several inactive, or very weakly active, compounds; two of which were identified as the iridoid glycosides, catalposide and specioside (weakly active). Dry fecal weight was the measure of feeding. El-Naggar, S. F. and Doskotch, R. W., J. Nat. Prod. 43: 524-527 (1980).
In 1915, researchers first observed that the green ash tree (Fraxinus pennsylvanica, family Oleaceae) had an antibiotic effect on L. dispar. Antibiosis was observed when the researchers examined the effects of F. pennsylvanica foliage on first- to fourth-instar larvae. Larvae placed on green ash leaves starting in any of the four stadia were unable to pupate. Mosher, F. H., USDA Technical Bulletin 250:3-39 (1915).
Later researchers found that second-instar L. dispar larvae died when an extract of neem seeds (Azadirachta indica), which contains azadirachtin (a known insect antifeedant and antibiotic), was added to an artificial diet. Skatulla, U. and Meisner, J., Umweltschutz 48: 38-40 (1975).
Other researchers have tested the effects of terpenoid-based compounds on L. dispar growth and survival. For example, when the four biosynthetically related iridoid (cyclopentan-(c)-pyran monoterpenoid) glycosides aucubin, catalpol, loganin and asperuloside, were added to an artificial diet, the researchers observed a significant reduction in larval growth and survival as compared to the control. Bowers, D. M. and Puttick, G. M., J. Chem. Ecol. 14: 319-333 1988.
The same researchers later tested the effect of different concentrations of the iridoid glycoside catalposide on the growth rate and the survival of two wild, and one laboratory, strains of larvae. Using much higher doses of catalposide than were used by El-Naggar and Doskotch (as reported in J. Nat. Prod. 43: 524-527, 1980), Bowers and Puttick found that one wild strain both grew (based on larval weight) and survived significantly better on the lowest allelochemical concentration. Growth rate and survival of the other two strains were not affected by treatments. Bowers, D. M. and Puttick, G. M., Ecol. Entomol. 14: 247-256 (1989).
Terpenoid-based compounds are prevalent in the family Oleaceae, which includes the green ash tree (genus Fraxinus). The potent insect deterrents iridoid glycosides (described above) are abundant in Oleaceae. The Oleaceae iridoid glycosides include syringenone and syringoxide (from genus Syringa and Phyllinea), syringopicroside (from genus Syringa) and forsythide and forsythide methyl ester (from genus Forsythia).
Monoterpenoid alcohols reported in the Oleaceae family include geraniol, d-linalool and 1-.alpha.-terpineol (found in genus Jasminum). Triterpenoids include oleanolic acid (from genus Ligustrum and genus Olea), O-acetyl-oleanolic acid (found in genus Ligustrum), squalene (from genus Olea) and ursolic acid (from genus Osmanthus). This family also contains the following tetraterpenoids: antheraxanthin esters, cryptoxanthin-epoxide esters, cryptoxanthin esters, flavoxanthin, lutein esters, neoxanthin esters and violaxanthin (from genus Forsythia), as reported in Gibbs, 1974, cited above. Secoiridoid glucosides found in the Oleaceae family, but not generally in the genus Fraxinus, include 10-hydroxy-ligustroside (from genus Ligustrum), 10-acetoxy-ligustroside and 10-acetoxy-oleuropein (from genus Osmanthus, as reported in El-Naggar and Beal, J. Nat. Prod. 3: 649-707, 1980), multifloroside, multiroside and 10-hydroxyoleoside-11- methyl ester (genus Jasminum, as reported in Shen et al., Phytochem. 29: 2905-2912, 1990), and 2" hydroxyjasminin, isojasminin, 4"-hydroxyisojasminin and jasmosidic acid (from genus Jasminum, as reported in Inoue et al., Phytochem. 30: 1191-1201, 1991).
Secoiridoids are known to be potent antifeedants against some insects. For example, xylomollin, a bitter compound of unripened fruits of Xylocarpus molluscensis, showed antifeedant activity against the African army worm. Kubo et al., J. Am. Chem. Soc. 98: 6704-6705 (1976). Secoiridoid glucosides specifically found in the genus Fraxinus include oleuropein (as reported in Inouye et al., Phytochemistry 14: 304, 1975); ligstroside and n uzhenide (as reported in LaLonde et al., J. Am. Chem. Soc. 98: 3007-3013, 1976); and neooleuropein (as reported in Kuwajima et al., Phytochemistry 31: 1227-1280, 1992). Kuwajima et al. also reported a new substance, frachinoside (cichoriinylsecoxyloganin), the first secoiridoid glucoside linked to a coumarin glucoside in the Fraxinus genus. LaLonde et al. (1976) also reported bisglycosidic secoiridoids GI-3 and GI-5 from Fraxinus spp.
The chemistry of the genus Fraxinus also includes numerous coumarin glycosides and various aglycones. A glycoside is a compound with a semiacetal linkage between a sugar and an alcoholic or phenolic group, which is called an aglycone. Paris, Chemical Plant Taxonomy, T. Swain [ed.]. Academic Press, London, pp. 337-358 (1963).
Coumarin glycosides in the genus Fraxinus include fraxin (as reported in Jensen et al., The Chemistry of Wood, B. L. Browning [ed.], John Wiley & Sons, New York, pp. 589-666, 1963; and Paris, 1963), esculin (as reported in Jensen et al., 1963; and Paris, 1963), cichorin (as reported in Harborne and Simmonds, Biochemistry of Phenolic Compounds, J. B. Harborne [ed.]. Academic Press, London, pp. 78-127, 1964) and daphnin (as reported in Robinson, The Organic constituents of higher plants. Their Chemistry and Interrelationships, 6th ed. Cordus Press, Massachusetts, 1991).
Coumarin aglycones in Fraxinus include fraxetin and isofraxetin (Harborne and Simmonds, 1964); esculetin (Robinson, 1991); and umbelliferone, scopoletin and xanthoxyletin (Jensen et al., 1963). These aglycones are reported to be strong antifeedants and antibiotics. Ngadjui et al., J. Nat. Prod. 52: 243-247 (1989); Su and Horvat, J. Agric. Food Chem. 35: 509-511 (1987).
Glycosides of flavonoids in Fraxinus include quercetin 3-O-rutinoside, luteolin 7-O-glucoside, luteolin 7-O-rutinoside, apigenin 7-O-rutinoside and acacetin 7-O-glucoside. The flavonoid aglycones, luteolin and 7-methoxy luteolin, were reported in green ash foliage. Black-Schaefer and Beckmann, Castanea 54: 115-118 (1989).